Socialization causes long-lasting behavioral changes

In modern human societies, social isolation acts as a negative factor for health and life quality. On the other hand, social interaction also has profound effects on animal and human, impacting aggressiveness, feeding and sleep, among many other behaviors. Here, we observe that in the fly Drosophila melanogaster these behavioral changes long-last even after social interaction has ceased, suggesting that the socialization experience triggers behavioral plasticity. These modified behaviors maintain similar levels for 24 h and persist up to 72 h, although showing a progressive decay. We also find that impairing long-term memory mechanisms either genetically or by anesthesia abolishes the expected behavioral changes in response to social interaction. Furthermore, we show that socialization increases CREB-dependent neuronal activity and synaptic plasticity in the mushroom body, the main insect memory center analogous to mammalian hippocampus. We propose that social interaction triggers socialization awareness, understood as long-lasting changes in behavior caused by experience with mechanistic similarities to long-term memory formation. Supplementary Information The online version contains supplementary material available at 10.1038/s41598-024-73218-w.


LONG-TERM SOCIALIZATION-INDUCED FEEDING BEHAVIOR REQUIRES cAMP SIGNALING
Flies that experienced social interaction show reduced food consumption when compared with flies that were socially reared and posteriorly isolated 16 .We used single-fly CApillary FEeding -sCAFEassay (modified from 17 ) to extend these findings.We compared grouped flies with animals singly reared since eclosion, meaning that they were socially naive.As expected, there was a significant decrease in food uptake of 5-day socialized flies when compared to individual flies in the immediate 24 hours (0 h-24 h time window) (fig 1A).Next, to determine if such feeding effect is maintained even in the absence of social interaction, we slightly modified the socially-enriched paradigm: flies were group-or single-reared for 5 days and then animals from both experimental groups were kept isolated for additional 24 hours previous to assessing feeding (fig 1B).Using this protocol, we also detected a decreased food consumption of grouped flies in the 24 h-48 h time window, confirming a long-lasting effect of social interaction on feeding behavior (fig 1C).We reasoned that the most plausible candidate genes to play a role for such long-lasting effect would be memory-related genes, such as rutabaga (rut) 18 .Despite their past experience, isolated rut mutant flies in the 24 h-48 h period after socialization showed no differences in food intake with solitaire animals since eclosion (fig 1C).Besides, rut mutant flies do not change their feeding behavior during the first 24 hours (0 h-24 h), suggesting a requirement of cAMP for this response (fig 1A).To confirm the involvement of cAMP signaling we repeated the sCAFE assay in animals mutant for dunce (dnc).
Results were comparable to rut mutant: dnc mutant flies fail to change their feeding behavior not only during the first 24 hours after socialization (0 h-24 h) but also according to previous experience, in the 24 h-48 h period (fig 1A , C).We propose that this long-lasting effect of social interaction on (feeding) behavior indicates a that animals have a socialization awareness that lasts even after social interaction has ceased.recently eclosed animals were either grouped or isolated for five days, and subsequently isolated for additional 24 hours before tested.C) Quantification of food consumption of wt, rut and dnc mutant flies in socialized and isolated conditions (sCAFE) in the 24 h-48 h h time window (Kruskal-

LONG-TERM EFFECT OF SOCIALIZATION ON SLEEP IN ABSENCE OF INTERACTION
Sleep (particularly daytime sleep) is also regulated after social interaction.When flies are kept in mixed-sex groups immediately at eclosion, sleep increases 14 .However, courtship experience inhibits sleep in male flies 19,20 , which lasts for several hours, proving a complex regulation of sleep by social cues and experiences.Most published sleep studies employ Drosophila Activity Monitors (DAMs), which only detect movement when the fly crosses a midpoint sensor in the housing tube 21 , overestimating actual sleep time 16 .The ethoscope was developed to unequivocally identify immobility periods and assess sleep 22 .We confirmed that social interaction also induced animals to sleep more We wondered how long animals that experienced a socially-enriched environment maintained such sleep behavior in absence of social interaction.To compare animals of the same age, we socialized flies for 7, 6 or 4 days (which is enough socialization time in order to generate a sleep effect 14 ), subsequently isolated them for 0, 1 or 3 days (named as socialized, 6+1, 4+3 or constant isolation).
Then, their sleep behavior was recorded for the following 3 days (fig 2D).In the framework of this experimental approach, we could compare continuously isolated flies with animals isolated for 1 to

days after socialization (fig 2E
).We could observe a progressive reduction of sleep time in the ZT0-ZT4 after isolation, with significant decrease at day 4 that was comparable to continuous isolation (fig 2F).Thus, 4 days of isolation are enough to modify sleep reaching similarly sleep levels than socially naive flies, in contrast to the need of 5 days described previously using DAMs 16 .
The ethoscope also allows a detailed sleep analysis regarding bout length, the total number of bouts and the latency to first bout.There were no differences in the sleep bout length amongst experimental groups.In contrast, isolated flies for 4 days reduced the number of sleep bouts to similar levels than the ones from socially naive animals, despite we noticed a progressive reduction but still statistically significative (fig 2H).Intriguingly, the latency to the first bout in grouped flies remained similar up to day 3, where it raised sharply, similar to the latency of isolated flies (fig 2I).
We conclude that the effect of socialization lasts at least for 3 days, and indeed, it can be considered as long-term.

SOCIALIZATION-INDUCED DIMINISHED AGGRESSIVENESS DEMANDS cAMP SIGNALING
In isolated flies that previously experienced social interaction, isolation signals starvation and, as a consequence, increases feeding and decreases sleep, meaning that both behavioral changes are reciprocally related 16 .We wondered if socialization awareness was also evident in a different social behavior.Previous data showed that 5-day grouped male flies since eclosion were less aggressive than their single-reared counterparts when tested immediately after the treatment 23 .This behavior also allowed us to determine the progression of long-lasting effects, in order to compare the temporal requirements of social interaction with those of classical learning and memory assays.
Thus, we employed animals from both experimental conditions and then evaluated aggression in a well-established paradigm after different isolation periods 24 .Socially-experienced flies showed reduced aggression at 1, 4, 8 and 24 hours after isolation when compared with single-reared animals (fig 3), evidencing a behavioral change at short-and long-term.Despite social interaction had ceased up to 24 h before, grouped flies still spent considerably less time fighting than lonely flies (fig 3), confirming that socialization awareness is a general feature of socialization.Critically, rut mutant flies did not show any difference according to their previous experience (fig 3), reinforcing the role of memory-related genes.Besides, our data revealed that at short-term (one hour), grouped rut mutant animals were significantly less aggressive than rut single-reared flies, thus differentiating from rut requirements in classical learning assays.However, this rutindependent effect disappeared after four hours, suggesting that social interaction imprinted a temporary effect independently of cAMP, but long-term socialization consequences in aggression depends on rut activity.

ANESTHESIA ABOLISHES SOCIALIZATION EFFECTS
Anesthesia blocks long-term memory consolidation in most species 25,26 4B).Given the reciprocal relationship between feeding and sleep behavior regarding social interaction 16 , we confirmed that sleep between lonely and socialized animals in the 24 h-28 h time window also remained similar after cold shock (fig 4C , D).
As expected, in non-shocked animals the difference was significant (fig 4C , D).In summary, we found that socialization awareness relies on cAMP signaling and is blocked by anesthesia, as it occurs in long-term memory.

PLASTICITY
In Drosophila, LTM increased the number of CREB-activated neurons in the MB 9,28 .To evaluate whether or not socialization also correlates with higher levels of CREB activity in the MB, we used the CAMEL reporter tool after 5 days of socialization directly after eclosion.This tool bears a MBspecific transgenic construct that responds to phosphorylated CREB with the production of GFP 28 .
We   LTM formation using an appetitive conditioning paradigm increased the number of MB-input synapses 29 .Thus, to determine if CREB-activated neurons after socialization also showed signals of increased synaptic plasticity, we included in the CAMEL tool a second reporter, the presynaptic marker BRP, fused with the RFP-variant cherry.This reporter combination allowed the visualization of the presynaptic densities (fig 5C).We quantified the number of synapses per cell volume in brains of 5-day grouped and single-reared animals (fig S2 shows an example of this quantification technique, see M&M).There was a significant increase in the relative number of pre-synapses in the MB of grouped flies compared to single-reared animals (fig 5D), similar to the synaptic plasticity described in mammals after a memorable experience 30 .In contrast, in a rut mutant background we could not detect any difference in the number of MB pre-synapses, which was in agreement with the reduced pre-synapse number in rut MB-input neurons after appetitive conditioning 29

(fig 5D).
Given that intensity of fluorescence varies greatly depending on the region, for analytical purposes we divided the MB in three areas, alpha, beta and the tip of beta.Interestingly, the former two showed only a marginal increase that did not reach statistical significance, however the tip of the MB concentrated most of the increase (fig S3).In summary, results showed a clear correlation of CREB-activated neurons and increased synaptic plasticity with effective social interaction that is abolished in memory impaired mutants, thus supporting a resemblance between socialization awareness and LTM.

DISCUSSION
Socialization induces several changes in animal behavior and here we show that such changes are long-lasting, as a result of social interaction experience.Not surprisingly, socialization awareness shows similarities with a long-term memory process: involvement of cAMP signaling and processes of neuronal and synaptic plasticity.However, it presents differences with LTM.For instance, the role of rut in short-term behavioral changes seems dispensable, at least for aggression (fig 3A , B).It may indicate that short-term effect is independent of rut.A striking peculiarity is its temporal dynamics since it would be hard to distinguish putative learning and consolidation stages during socialization, while in long-term memory paradigms both phases are clearly distinguishable (as, for example, in appetitive or aversive olfactory conditioning).
The classic view on sleep regulation indicates that this behavioral state is regulated by the circadian clock and the internal sleep homeostat 31 , but recent work in many species including Drosophila show that sleep regulation goes beyond these two processes and includes temperature, starvation, sexual arousal, and social context, among others 32 .Our data suggest that recalling a past social experience may also regulate sleep in flies (fig 1), similar to what happens with psychophysiologic insomnia in humans 33 .
In mammals, social isolation has profound effects on behavior and cognition, which is accompanied by detectable alterations in brain structure and function at several levels 34 .For instance, the hyppocampus shows reduced dendritic spine density after either postnatal or juvenile social isolation 35,36 .The hyppocampus is the main structure related to long-term memory, analog to the insect Mushroom Body 37 .In fact, it was previously described that socialization increases the fiber number in the MB, an increase that is impeded by classic learning mutation such as rutabaga 12,13 .
In addition, our results revealed that socialization also induces rut-dependent changes in synaptic plasticity of the previously activated MB neurons.The increased synaptic densities in CREBpositive neurons might be explained by the socialization-induced enhanced sleep, given that sleep loss diminishes pre-synaptic densities in cholinergic neurons, including the MB neurons 38,39 .This is unlikely because despite rut mutant animals do sleep much more (fig 2B), their MB presynaptic densities do not reach levels of socialized wt animals, although they are higher than in isolated flies (fig 5D).Besides, rut mutant flies did not reach enough sleep levels as to restore behavioral plasticity 40 , thus suggesting that rut increased synaptic activity might be due to the excess of sleep but it is unable to rescue the effect of social interaction (fig 1 -3).This reinforces the idea that socialization awareness may induce behavioral plasticity by similar mechanisms to long-term memory.
Why do isolated flies that were previously socialized behave similar to single-reared flies since eclosion 14 ?Actually, chronic isolation displays starvation-like phenotypes in Drosophila 16 and starvation disables aversive long-term memory 41 , probably because increased metabolism in the MB and glia are necessary 42,43 .It might well be that socialization awareness was prevented as a consequence of the starvation signaling, and this would explain the similar phenotypes achieved by isolation after socialization and isolation since eclosion, despite mechanistically they should be different.Indeed, one might hypothesize that rescuing such starvation-like phenotype would reveal differences between both experimental conditions.Notably, socialization-induced behavioral changes are sexually dimorphic, since grouped and single-reared females behave similarly 44 .Indeed, male-specific P1 interneurons act as an internal state regulatory hub for sleep, aggression, sleep and spontaneous 45 .Together with Diuretic hormone 44-(DH44) and Tachykinin-(TK) expressing interneurons, P1 neurons form a malespecific neural circuit that regulates spontaneous locomotion in response to social interaction, thus suggesting a possible common mechanism for socially-induced behavioral changes 44 .
Interestingly, P1 neurons directly activate a specific subset of dopaminergic neurons that innervate the MB and it drives LTM appetitive olfactory memory formation 46 .The MB is not only a memory regulatory center but also acts as a sleep and feeding regulatory center 47,48 .In this work we have shown that social interaction correlates with increased synaptic plasticity in the MB itself (fig 4).Thus, it is interesting to speculate that socialization awareness may use a general neural circuit connecting P1 neurons, dopaminergic neurons and the MB in order to modify several behaviors with long-lasting effects.

Materials -Stocks and fly husbandry
Flies were raised and experiment performed using standard food at 25ºC on a 12/12h light/dark cycle.rutabaga 2080 (#9405), dunce 1 (#6020) and Wild Type (Canton S #64349) stocks were obtained from Bloomington Drosophila Stock Center.The CAMEL tool is composed by 6xCRE-splitGal4 AD , UAS-eGFP and R21B06-splitGal4 DBD , gently donated by Dr Jan Pielage 28 .rut 2080 ; 6xCRE-splitGal4 AD and UAS-cherry-Brutchpilot; R21B06-splitGal4 DBD stocks were combined in our laboratory and are available under request.

-Isolation/socialization Protocol
Male virgin flies were collected under CO2 anesthesia within 4 hours post-eclosion and isolated in individual glass vials or socialized (25:25 male:female) in a plastic bottle.After 5 days of socialization or isolation, all flies were isolated without using anesthesia for 24 h (except where indicated) and then, behavioral experiments or dissections were performed.
In the case of cold shock, flies were ice-cold shocked twice a day (Zeitgeber Time 1 -ZT01-and ZT9) during the five days of isolation/socialization protocol for 2-3 minutes (i.e. until flies fainted).
Glass vials were used to allow good cold transfer from ice.Afterwards vials were placed horizontally in a RT surface to let flies recover.

-single fly Capillary Feeding (sCAFE)
The protocol from 17 was used with slight modifications.Males were placed in individual vials with a wet filter paper at the bottom and a 5 µl capillary (Blaubrand, 708707) with 5% sucrose water food.The capillary was introduced through a 5mm cut 200 µl pipet tip that goes through a wet plug and sustained with an additional tip.After 24 h food intake is measured (0 h-24 h time window), the capillary substituted by a new one and plugs are wet again to preserve moisture.24 h later food intake is measured again.Once the experiment has finished flies are weighted.Additional 3 individual tubes without flies were measured to control the evaporation rate.

-Sleep
For all experiments, flies were sorted into glass tubes [70 mm × 5 mm × 3 mm (length × external diameter × internal diameter)] containing the same food used for rearing under a regime of 12:12 Light:Dark (LD) condition in incubators set at 25°C.Activity recordings were performed using ethoscopes 22 .Behavioural data analysis was performed in RStudio (RStudio Team.RStudio: Integrated Development for r.RSudio, Inc. Boston, MA; 2015.http://www.rstudio.com/)employing the Rethomics suite of packages 49 .All sleep assays were repeated at least twice with 20-40 flies/treatment/experiment.

-Aggression
The protocol from 24 with slight modifications was used.Briefly, two flies were placed into each chamber of the arena (4x3 mm grid) with food.One-to-one socialization was achieved by allowing both flies to interact, whereas isolation was caused by a black divider that allowed physical separation of flies.After 5 days, socialized flies were also separated by the divider for 1, 4, 8 or 24 h.After removing the divider, reunited flies were recorded for 20 minutes and agression analyzed by means of the FlyTracker (MATLAB) software and the platform JAABA (Janelia Automatic Animal Behavior Annotator), that identifies when the animal is lunging.The proportion of time fighting is the number of frames in which a particular animal lunges divided by the total number of frames.

-Immunolabeling, imaging and image analysis
Adult brain preparations were stained following the same protocol as in 50 .Dissections were always performed at ZT4-5 to avoid possible circadian-induced changes.
Images were taken by a Leica SP5 confocal microscopy re-using the same experimental conditions, avoiding saturation.CREB+ cell images were taken using a 40X objective, with slices of 3 um.Synapse quantification confocal images were taken the same day using a 63X objective, slices of 0,8um.Posteriorly images were treated using Imaris 6.3.1 software.Axon volume was rebuilt using the Volume tool and brutchpilot signal was quantified using the Spots tool.To adjust brightness parameters accurately the MB was divided in three parts (alfa, beta and beta tip) (Fig.

-Statistical analysis
For the behavioral and morphological experiments (figures 1, 2, 3, 4, 5, S1 and S3), the data was analyzed in R (version 3.6.3)through Rstudio (Version 1.0.153),employing the Kruskal-Wallis nonparametric test (library stats).When appropriate, we performed post hoc Dunn analyses (library FSA) to identify specific differences between treatments.All assays were repeated at least twice with sample sizes as indicated within the figure

Figure 1 .
Figure 1.Reduced food consumption induced by socialization depends on memory-related Figure 1 -Gil M

Figure 3 .
Figure 3. Socialized-reduced aggression shows short-and long-term effect.Quantification of

Fig 4 .
Fig 4. Anesthesia abolishes socialization effects on sleep and food consumption.(A)

Feeding
INCREASED NEURONAL ACTIVITY AND SYNAPTIC quantified the number of GFP positive soma (fig 5A) in adult brains, observing an increase in the number of CREB-positive cells in grouped vs single-reared animals (fig 5B).In contrast, this CREB response was lost in rut mutant brains (fig 5B).

Figure 5 .
Figure 5. Socialization correlates with cellular and synaptic plasticity.A) Representative

Supp. 2 )
. Synaptic density for each Mushroom Body is the summatory of spots/volume from each part.

Figure 2. Sleep behavior after socialization is long-term and relies on rut.
14,16g2A).To ensure consistency with previous works16, we focused on the first four hours after lights ON, where the effect is unambiguous and reproducible (i.e.248 h time window, ZT0-ZT4, fig2A).Sleep quantification shows a significant difference between socialenriched and lonely animals in this 24 h-28 h period (fig 2B), in line with previous publications14,16.(G),total number of bouts (H) and latency to first bout (I) from ZT0 to ZT4.
27gressionolfactory conditioning assay27.We wondered if anesthesia was also able to block socialization awareness.We exposed adult flies to 3-min cold shock two times per day to single and grouped flies during the training period (fig4A).Both experimental "cold-shocked" groups did not show any significant differences in food consumption in the 24 h-48 h time window after isolation, in contrast to non-shocked control animals (fig . In Drosophila, a 2-min cold shock acts as anesthetics and is able to impede long-term memory in the classical aversive